Translocation-independent activation of protein kinase C by platelet-activating factor, thrombin and prostacyclin. Lack of correlation with polyphosphoinositide hydrolysis in rabbit platelets

Abstract

The relationship between polyphosphoinositide hydrolysis and protein kinase C (PKC) activation was explored in rabbit platelets treated with the agonists platelet-activating factor (PAF), thrombin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and with the anti-aggregant prostacyclin (PGI2). Measurement of the hydrolysis of radiolabelled inositolcontaining phospholipids relied upon the separation of the products [3H]inositol mono-, bis-and tris-phosphates by Dowex-l chromatography. PKC activity, measured in platelet cytosolic and Nonidet-P40-solubilized particulate extracts that were fractionated by MonoQ chromatography, was based upon the ability of the enzyme to phosphorylate either histone HI in the presence of the activators Ca2+, diacylglycerol and phosphatidylserine, or protamine in the absence of Ca2+ and lipid. Treatment of platelets for 1 min with PAF (2 nM) or thrombin (2 units/ml) led to the rapid hydrolysis of inositol-containing phospholipids, a 2-3-fold stimulation of both cytosolic and particulate-derived PKC activity, and platelet aggregation. Exposure to TPA (200 nM) for 5 min did not stimulate formation of phosphoinositides, but translocated more than 95 % of cytosolic PKC into the particulate fraction, and induced a slower rate of aggregation. PGI2 (1 ,ug/ml) did not enhance phosphoinositide production, and at higher concentrations (50 ,ug/ml) it antagonized the ability of PAF, but not that of thrombin, to induce inositol phospholipid turnover, even though platelet aggregation in response to both agonists was blocked by PGI2. On the other hand, PGI2 alone also appeared to activate (by 3-5-fold) cytosolic and particulate PKC by a translocation-independent mechanism. The activation of PKC by PGI2 was probably mediated via cyclic AMP (cAMP), as this effect was mimicked by the cAMP analogue 8-chlorophenylthio-cAMP. It is concluded that this novel mechanism of PKC regulation by platelet agonists may operate independently of polyphosphoinositide turnover, and that activation of cAMP-dependent protein kinase represents another route leading to PKC activation.

Erbstatin blocks platelet activating factor‐induced protein‐tyrosine phosphorylation, polyphosphoinositide hydrolysis, protein kinase C activation, serotonin secretion and aggregation of rabbit platelets

“…Within 1 min of either PAF or thrombin exposure to rabbit platelets, both the cytosolic and 1% NP40-solubilized particulate PKC activities become elevated 2-3-fold as measured in MonoQ-fractionated extracts [5,8]. Regardless of whether the Ca", diacylglycerol and phosphatidyl- Erbstatin cont.…”

“…The elevation of Ca2+ and,diacylglycerol has been linked with the activation of a family of Ca2+-activated, phospholipid-dependent protein kinase (PKC) isozymes [7]. Treatment of rabbit platelets with PAF leads to 2-3-fold increases in both the activities of cytosolic and membrane-bound forms of PKC within one minute [5,8]. The pivotal role that this kinase plays in platelet activation by PAF and other cytokines is implied by the ability of a range of PKC inhibitors to block such downstream responses to these agonists as cell aggregation and the release of mediators like serotonin [9-121. Within seconds of exposure to human platelets to thrombin and collagen, enhanced tyrosine …”

Studies on the role of protein kinases in the TNF‐mediated enhancement of murine tumor cell‐endothelial cell interactions

“…However, there is increasing evidence showing the lack of correlation between translocation and biological effects of PKC in different cells (Bosca et al, 1989). For example, Salari et al (1990) showed that platelet activating factor, thrombin, and prostacyclin caused stimulation of both cytosolic and particulate-derived PKC in rabbit platelets. Moreover, IL-3 appeared to activate the preexisting membrane-associated PKC rather than elicit its translocation from the cytoplasm in a mast cell/megakaryocyte line R6-XE.4 (Pelech et al, 1990).…”

The level of expression of a protein kinase C gene may be an important component of the patterning process in Hydra

“…Since the amount of PKC as well as expression of HvPKC2 also increases in foot-regenerating fragments, and since it is unlikely that HvPKC2 should not be used for signalling there, other mechanisms must be postulated to account for PKC activation during foot formation. DAG and other PKC-activating lipids are also generated, for example, by agonist-induced hydrolysis of choline-phospholipids (Naor 1991;Nishizuka 1992), and cytosolic PKC can be activated without translocation or remain active after a short initial translocation (Salari et al 1990;Trilivas et al 1991). It remains to be clarified, if these alternative pathways are used by foot-forming tissue to activate PKC.…”